Identification of monellin as the first naturally derived proteinaceous allosteric agonist of metabotropic glutamate receptor 5

Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of "selective" allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu₅ ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu₅ . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu₅ demonstrated that monellin agonism was not mediated via the "common" allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu₅ . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu₅ PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu₅ agonism was positively modulated by the mGlu₅ pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu₅ , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.